p53 acts as a tumor suppressor protein, which participates in cell growth and apoptosis. Mutation of the p53 gene brings about inactivation of its translation product, which is a major cause of cancer. However, in some cases, although the p53 gene is present in the wild form, its protein product is often inactivated. One of such p53 inactivation mechanisms involves the overexpression of mdm2, a cellular oncogene. The p53 protein is autoregulated by a feedback loop mediated by the mdm2 protein, inhibiting p53 function through binding to the transcriptional activation domain (TAD) of p53, and the mdm2 protein binds to p53 and promotes p53 degradation through the ubiquitin-dependent pathway, thereby promoting the incidence of cancer (Oliner, J. D. et al. (1993) Nature 362, 857; Vassilev, L. T. et al. (2004) Science 303, 844-848; and Maki et al. (1999) J. Biol. Chem. 274, 1 6531).
Based on the finding that the mdm2 protein is amplified or overexpressed in many malignant tumors, leading to the inhibition of p53 function, the activation of the p53 pathway through the inhibition of mdm2 is becoming a new target for developing caner therapeutic agents.
One strategy to do this involves the development of low molecular weight agents capable of inhibiting the complex formation of p53 and mdm2. For example, piperazine derivatives useful in cancer therapy by inhibiting the interaction between mdm2 and p53 are disclosed in WO00/15657, U.S. Pat. No. 6,770,627, etc. WO03/051360 discloses the use of cis-imidazolines as mdm2 inhibitors, which are useful for the treatment of breast cancer, large intestine cancer, lung cancer, and the like. WO03/095625 discloses 1,4-benzodiazepines that act as inhibitors against the interaction between mdm2 and p53. WO03/106384A2 discloses boronic chalcone derivatives that inhibit mdm2 expression or complex formation with mdm2. Also, Stoll et al. describes chalcone derivatives inhibiting the binding of mdm2 to p53 (Stoll, R. et al. (2001) Biochemistry 40, 336-344).
Another strategy involves the development of mdm2-specific antisense oligonucleotides that inhibit mdm2 protein expression. Many publications, including WO99/49065 and WO99/10486, U.S. Pat. Nos. 6,013,786 and 6,238,921, and European Pat. No. 1 007 658, provide such antisense oligonucleotides.
In addition, other studies describe peptides that specifically bind to mdm2 and inhibit the binding of mdm2 to p53. The X-ray crystal structure of mdm2 bound to the p53 TAD peptide reveals that only a helix formed by residues 18-26 of the p53 TAD binds to mdm2 (Kussie, R. H. et al., (1996) Science 274, 948-953). Since then, based on this X-ray crystal structure, many studies have been focused on the p53 α-helix. For example, WO96/02642 provides a peptide encompassing amino acid residues 19-23 of the p53 α-helix. U.S. Pat. No. 5,702,908 provides a peptide encompassing amino acid residues 18-23 of the p53 α-helix. WO98/476525 provides a peptide encompassing amino acid residues 19-23 of the p53 α-helix. WO98/01467 provides a peptide encompassing amino acid residues 19-26 of the p53 α-helix. U.S. Pat. No. 5,858,976 provides a peptide encompassing amino acid residues 14-41 of the p53 α-helix. These peptides, consisting of amino acid residues of the p53 α-helix region, are characterized by inhibiting mdm2.
The present inventors, in a previous study, reported that, unlike the previous view that the p53 TAD is unstructured, the p53 TAD has local secondary structures such as an α-helix and a turn even in the state of being unbound to mdm2 (Lee, H. et al. (2000) J. Biol. Chem. 275, 29426-29432).
To date, all of studies involving peptides inhibiting the binding of mdm2 and p53 have been focused only on the helix region of the p53 TAD, based on the X-ray crystal structure of the complex of mdm2 with the p53 TAD peptide. Also, there is no research describing substantial binding between mdm2 and a turn that is another secondary structure of the p53 TAD, and turn-derived peptides inhibiting the binding of mdm2 and p53.
In this regard, the intensive and thorough research into the effect of the turns of the p53 TAD on mdm2 binding thereto by chemical shift perturbation experiments using nuclear magnetic resonance (NMR) spectroscopy resulted in the finding that the turns as well as the helix participate directly in the binding of the p53 TAD to mdm2, and in particular, a region encompassing amino acid residues 49-54, forming turn II, is most critical for the binding to mdm2. The finding further includes that, when a peptide sequence corresponding to this region or a derivative thereof is administered to cancer cells, it inhibits the binding of mdm2 to p53 and activates the p53 pathway to induce apoptosis, thereby leading to the present invention providing peptides for inhibiting mdm2.